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Title: Homologous recombination plays minor role in excision of unit-length viral genomes from head-to-tail direct tandem repeats of porcine circovirus during DNA replication in Escherichia coli

Author
item CHEUNG, ANDREW

Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2007
Publication Date: 8/1/2007
Citation: Cheung, A.K. 2007. Homologous recombination plays minor role in excision of unit-length viral genomes from head-to-tail direct tandem repeats of porcine circovirus during DNA replication in Escherichia coli. Archives of Virology. 152:1531-1539.

Interpretive Summary: Porcine circovirus type 2 (PCV2) remains an important viral pathogen of swine. While clinical signs of disease and postmortem lesions induced by PCV2 are known, there is little information on the temporal pathogenesis and epidemiology of the virus. In previous work, we examined the genetic elements synthesized by PCV type 1 (PCV1) and PCV type 2 (PCV2) in tissue culture cells. We have identified several new PCV2 genetic elements that are different from the non-pathogenic PCV1 and have determined the essential and non-essential genetic elements required for PCV1 and PCV2 replication. We also proposed a novel rolling circle "melting-pot" model to account for its replication. Recently, we discovered that PCV DNA replication can occur in E. coli via the rolling-circle mechanism. In this work, we examined the role of homologous recombinantion in the generation of unit-length viral genomes from head-to tail tandem repeat constructs. Thus, this work provides insight into the origin of PCV and a general framework to generate recombinant viruses more efficiently. The information obtained advances our understanding of circovirus biology and aids the research of scientists in industry, universities and government agencies.

Technical Abstract: Previously, we demonstrated that a theta-replicating bacterial plasmid containing 1.75 copies of genomic porcine circovirus (PCV) DNA in head-to-tail tandem (HTT) [a partial copy of PCV type 1 (PCV1), a complete copy of PCV type 2 (PCV2) and two origins of DNA replication (Ori)] yielded three different double-stranded DNA species when transformed into Escherichia coli: the input construct (U), the unit-length PCV1/PCV2 genome with a composite Ori lacking the plasmid vector (Q) and the remaining left-over 0.75 copy PCV1/PCV2 genome with a different composite Ori inserted in the plasmid vector (L). Replication of U was via the theta-like replication mechanism utilizing the colicin E1 Ori; while derivation of L and Q was via the rolling-circle replication (RCR) copy-release mechanism and requires the presence of two PCV Oris and the virus-encoded Rep protein. Essentially, excision of a unit-length PCV1/PCV2 genome via RCR from U yielded L and Q. In this work, we examined whether homologous recombination may also result in excision of unit-length PCV genomes from HTT constructs. The results showed that homologous recombination plays a minor role and the unit-length DNA species derived from homologous recombination possess different characteristics from those generated via the RCR mechanism.